1. Field of the Invention
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the 32P labeling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest.
Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient""s body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.
When very low concentrations must be detected, the above method is slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable.
A method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the hybridization sites on the DNA sequence complementary to the 5xe2x80x2 ends of the oligonucleotide primers.
Other methods for amplifying nucleic acids are single primer amplification, ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and the Q-beta-replicase method. Regardless of the amplification used, the amplified product must be detected.
Genetic recombination involves the exchange of DNA strands between two related DNA duplexes. The branch point between two duplex DNAs that have exchanged a pair of strands is thought to be an important intermediate in homologous recombination. This branch point is otherwise referred to as the Holliday junction. Movement of the Holliday junction by branch migration can increase or decrease the amount of genetic information exchanged between homologues. In vitro strand exchange is protein mediated, unlike the spontaneous migration that occurs in vitro.
There is a great demand for simple universal high-throughput methods for detection of differences in related nucleic acid sequences regardless of the exact nature of the difference. This demand is becoming more and more urgent due to the ongoing rapid discovery of new disease related mutations brought about by the progress of the Human Genome Project. A detection method for mutations that is not dependent on the exact location of the mutation is valuable in the case of diseases that are known to result from various mutations within a given sequence. Moreover, such a method will be useful for verification of sequence homology as related to various applications in molecular biology, molecular medicine and population genetics.
Some of the current methods are either targeted for sets of known mutations, such as, for example, the Reverse Dot Blot method, or involve gel-based techniques, such as, for example, single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) or direct sequencing as well as a number of methods for the detection of heteroduplexes. Accordingly, such methods are laborious and time consuming.
Various methods for mutation detection have been developed in the recent years based on amplification technology. The detection of sequence alterations is based on one of the following principles: allele-specific hybridization, chemical modification of mismatched bases with subsequent strand cleavage, nuclease cleavage at mismatches, recognition of mismatches by specific DNA binding proteins, changes in electrophoretic mobility of mismatched duplexes in gradients of denaturing agents, conformation-induced changes in electrophoretic mobility of single-stranded DNA sometimes combined with conformation-specific nuclease cleavage. Some of these methods are too laborious and time-consuming and many depend on the nature of base alteration.
It is desirable to have a sensitive, simple, inexpensive method for detecting differences in nucleic acids such as mutations, preferably, in a homogeneous format. The method should minimize the number and complexity of steps and reagents. Such a method would be suitable for a large scale population screening.
2. Description of the Related Art
Formation of a single base mismatch that impedes spontaneous DNA branch migration is described by Panyutin, et al., (1993) J. Mol. Biol., 230:413-424.
The kinetics of spontaneous DNA branch migration is discussed by Panyutin, et al., (1994) Proc. Natl. Acad. Sci. USA, 91: 2021-2025.
European Patent Application No. 0 450 370 A1 (Wetmur, et al.,) discloses branch migration of nucleotides.
A displacement polynucleotide assay method and polynucleotide complex reagent therefor is discussed in U.S. Pat. No. 4,766,062 (Diamond, et al.,).
A strand displacement assay and complex useful therefor is discussed in PCT application WO 94/06937 (Eadie, et al.,).
PCT application WO/86/06412 (Fritsch, et al.,) discusses process and nucleic acid construct for producing reagent complexes useful in determining target nucleotide sequences.
A process for amplifying, detecting and/or cloning nucleic acid sequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 and 5,008,182. Sequence polymerization by polymerase chain reaction is described by Saiki, et al., (1986) Science, 230: 1350-1354. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase is described by Saiki, et al, Science (1988) 239:487.
One method in accordance with the present invention is directed to the detection of the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both of the nucleic acid sequences in double stranded form. Both members of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of the difference between the two related sequences. The complex may comprise a Holliday junction. The complex does not dissociate at least until detection of the labels as part of the complex has taken place and therefore, is in that sense a stable complex.
Another embodiment of the present invention is a method for detecting the presence of a difference between two related nucleic acid sequences. A medium suspected of containing two related nucleic acid sequences is treated to provide two partial duplexes each comprised of fully matched duplexes having at one end non-complementary end portions. The partial duplexes are related in that, except for the difference, one of the strands S1 of one of the partial duplexes is complementary to one of the strands S1xe2x80x2 of the other of the partial duplexes and the other of the strands S2 of one of the partial duplexes is complementary to the other of the strands S2xe2x80x2 of the other of the partial duplexes. The medium is subjected to conditions that permit the binding of S1 to S1xe2x80x2 and S2 to S2xe2x80x2, respectively. If the medium contains a difference between the related nucleic acid sequences, a stable complex is formed comprising strands S1, S1xe2x80x2, S2 and S2xe2x80x2. A determination is made whether the stable complex is formed, the presence thereof indicating the presence of a difference between the related nucleic acid sequences.
Another aspect of the present invention is a method for detecting a mutation within a target nucleic acid sequence. The method comprises forming from the target sequence a tailed target partial duplex comprising a duplex of the target sequence, a label and at one end of the duplex, two non-complementary oligonucleotides, one linked to each strand. The tailed target partial duplex is provided in combination with a labeled tailed reference partial duplex lacking the mutation. The tailed reference partial duplex is comprised of two nucleic acid strands that are complementary to the strands in the tailed target partial duplex but for the possible presence of a mutation. Labels are present in non-complementary strands of the tailed target and tailed reference partial duplexes, respectively. The formation of a stable complex between the tailed partial duplexes is detected by means of the labels. The formation of the complex is directly related to the presence of the mutation.
Another aspect of the present invention is a method of detecting a mutation within a target nucleic acid sequence, which is first amplified by the polymerase chain reaction using primers P1 and P2 to produce an amplicon AA. At least one of the primers P1 and P2 contains a label and primer P1 is comprised of a 3xe2x80x2-end portion Pa that can hybridize with the target sequence and 5xe2x80x2-end portion B1 that cannot hybridize with the target sequence. A chain extension of a primer P3 along one strand of amplicon AA is carried out to produce a tailed target partial duplex Axe2x80x2. Primer P3 is comprised of 3xe2x80x2-end portion Pa and a 5xe2x80x2-end portion A1 that cannot hybridize to the target sequence. A reference nucleic acid sequence is also amplified, using primer P2 and primer P3, by polymerase chain reaction to produce amplicon BB. The reference sequence is identical to the target sequence but lacking a possible mutation. Generally, when primer P2 used in the amplification of the target nucleic acid sequence comprises a label, primer P2 used for amplification of the reference nucleic acid sequence comprises a label that may be the same as or different than the label of primer P2. When primer P1 used for amplification of the target nucleic acid sequence comprises a label, primer P3 comprises a label that may be the same as or different than the label of primer P1. A chain extension of primer P1 along one strand of amplicon BB is carried out to produce a tailed reference partial duplex Bxe2x80x2, which is allowed to bind to the tailed target partial duplex Axe2x80x2. The binding of one labeled strand to the other labeled strand as a result of the formation of a stable complex between the tailed partial duplexes is detected, the binding thereof being directly related to the presence of the mutation.
Another aspect of the present invention is a method for detecting a mutation in a nucleic acid, wherein a partial duplex Axe2x80x2 is produced from a target nucleic acid sequence suspected of having a mutation. Partial duplex Axe2x80x2 comprises a fully complementary double stranded nucleic acid sequence containing the target nucleic acid sequence. One strand thereof has at its 5xe2x80x2-end a portion A1 that does not hybridize with a corresponding portion A2 at the 3xe2x80x2-end of the other strand. At least one of the strands of the partial duplex Axe2x80x2 comprises a label. A partial duplex Bxe2x80x2 is produced from a reference nucleic acid sequence that corresponds to the target nucleic acid sequence except for the mutation. Partial duplex Bxe2x80x2 comprises the double stranded nucleic acid sequence lacking the mutation wherein the strand corresponding to the strand comprising the portion A1 has at its 5xe2x80x2-end a portion B1 that is complementary with A2 and the other strand has at its 3xe2x80x2-end a portion B2 that is complementary with A1. One of the strands of partial duplex Bxe2x80x2 comprises a label wherein such strand is unable to hybridize directly to the strand of partial duplex Axe2x80x2 that comprises a label. Partial duplexes Axe2x80x2 and Bxe2x80x2 are subjected to conditions that permit the duplexes to hybridize to each other. If the target nucleic acid sequence having the mutation is present, a stable complex is formed comprising partial duplex Axe2x80x2 and partial duplex Bxe2x80x2, the presence thereof indicating the presence of the nucleic acid having the mutation.
Another embodiment of the present invention is a method for detecting a mutation in a target nucleic acid. A medium containing (i) the target nucleic acid suspected of having the mutation and (ii) two primers P1 and P2, wherein P1 is extendable along one of the strands of the nucleic acid, is subjected to temperature cycling in the presence of a nucleotide polymerase and nucleoside triphosphates. P1 has a 3xe2x80x2-end portion Pa that does bind, and a 5xe2x80x2-end portion B1 that does not bind, to one of the target nucleic acid strands. P2 is extendable along the other of the strands of the target nucleic acid. In this way one extended primer is a template for the other of the primers. The medium is then combined with a primer P3, which has the 3xe2x80x2-end portion Pa and a 5xe2x80x2-end portion Al that does not bind to the extended P2 primer. The medium is then subjected to conditions such that P3 is extended along extended primer P2 to produce only a complementary strand, and not a copy, thereof. A medium containing (i) a reference nucleic acid, which has an identical sequence to the sequence of the target nucleic acid except for the mutation and (ii) two primers P3 and P2 is subjected to temperature cycling in the presence of a nucleotide polymerase and nucleoside triphosphates. P3 is extendable along one of the strands of the reference nucleic acid, wherein Pa binds, and A1 does not bind, thereto. P2 is extendable along the other of the strands of the reference nucleic acid. Extended primer produced by the extension of one of the primers is a template for the other of the primers. The latter medium is combined with primer P1 wherein Pa binds to, and B1 does not bind to, the extended primer produced by extending P2 along the reference nucleic acid. The latter medium is subjected to conditions under which P1 is extended along extended primer P2 to produce only a complementary strand, and not a copy, of the extended primer P2. The above steps may be carried out together in the same medium or in separate reaction media and may be carried out simultaneously or wholly or partially sequentially. If the reactions are carried out in separate reaction media, the media are combined and subjected to conditions that permit the complementary strands produced in the above steps to bind to the extended primers P1 and P3, respectively, such that a stable complex is formed if a mutation is present in the target nucleic acid. A determination is made as to whether such complex is formed, the presence thereof indicating the presence of the mutation.
Another aspect of the present invention concerns a method of preparing a DNA partial duplex having a portion at an end thereof that has two predefined non-complementary single stranded sequences. A medium containing a nucleic acid is combined with a polymerase, nucleoside triphosphates and two primers. One of the primers P3 is extendable along one of the strands of the nucleic acid. P3 has a 3xe2x80x2-end portion Pa that does bind, and a 5xe2x80x2-end portion A1 that does not bind, thereto. The other of the primers P2 is extendable along the other of the strands of the nucleic acid. Extended primer produced by the extension of one of the primers is a template for the other of the primers. The medium is subjected to temperature cycling to extend the primers and then combined with a primer P1, which has 3xe2x80x2-end portion Pa that binds, and a 5xe2x80x2-end portion B1 that does not bind, to the extended primers. The medium is subjected to conditions such that P3 binds to and is extended along the extended primer P2 to produce only a complement, and not a copy, of the extended primer.
Another aspect of the present invention is a method of preparing a DNA partial homoduplex having a portion at one end that has two non-complementary single stranded sequences. A medium containing a single stranded polynucleotide is combined with a primer P1 wherein P1 has a 3xe2x80x2-end portion Pa that binds to a sequence that is 8 to 60 nucleotides from the 3xe2x80x2-end of the single stranded polynucleotide and a 8 to 60 nucleotide portion B1 that does not bind to the single stranded polynucleotide. The medium is subjected to conditions under which P1 binds to and is extended along the single stranded polynucleotide.
Another aspect of the present invention is a method for detecting a difference between two related nucleic acid sequences. A stable quadramolecular complex is formed comprising both of the nucleic acid sequences in double stranded form. The presence of the stable complex is detected by binding the complex to a receptor. The presence of the stable complex indicates the presence of a difference between the two related sequences.
The present invention also includes kits for determining a target nucleic acid sequence. A kit in accordance with the present invention comprises in packaged combination (a) a primer P2 that is extendable along one of the strands of the target nucleic acid, (b) a primer P1 comprising a 3xe2x80x2-end portion Pa that binds to and is extendable along the other of the strands of the target nucleic acid and a 5xe2x80x2-end portion B1 that does not bind to the target nucleic acid, and (c) a primer P3 comprising 3xe2x80x2-end portion Pa and a portion A1 that is different than B1 and does not bind to the target nucleic acid. In one aspect, the above reagents can be packaged in the same container. The kit can further include a reference nucleic acid and also may include a polymerase, nucleoside triphosphates, and a pair of primers for amplifying both the target nucleic acid and the reference nucleic acid sequences in order to increase the number of molecules for the practice of the present invention particularly for detection of a mutation in a target nucleic acid sequence.
Another aspect of the present invention is a method for detecting a target nucleic acid sequence wherein a tailed target partial duplex Axe2x80x2 is formed from the target sequence and is comprised of a duplex of said target sequence, a label, and at one end of the duplex, two non-complementary oligonucleotides, one linked to each strand. A combination is provided comprising (i) the tailed target partial duplex Axe2x80x2 and (ii) a tailed reference partial duplex Bxe2x80x2, which comprises a duplex of a sequence different than the target sequence, a label and, at one end of the duplex, two oligonucleotides that are complementary to the two non-complementary oligonucleotides, one linked to each strand. The formation of a complex between the partial duplexes Axe2x80x2 and Bxe2x80x2 is detected by means of the labels, the formation thereof being directly related to the presence of the target nucleic acid sequence.
Another embodiment of the present invention is a method of detecting a target nucleic acid sequence. The method comprises amplification of the target sequence by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA. At least one of primers P1 and P2 comprises a label and primer P1 is comprised of a 3xe2x80x2-end portion Pa that can hybridize with the target sequence and 5xe2x80x2-end portion B1 that cannot hybridize with the target sequence. A primer P3 is extended by chain extension along one strand of amplicon AA to produce a tailed target partial duplex Axe2x80x2. Primer P3 is comprised of 3xe2x80x2-end portion Pa and a 5xe2x80x2-end portion A1 that cannot hybridize to the target sequence or its complement. A reference nucleic acid sequence different than the target nucleic acid sequence is amplified, using primer P2 and P3, by polymerase chain reaction to produce amplicon BB. When primer P2 used in the amplification of the target nucleic acid sequence comprises a label, primer P2 used for the amplification of the reference nucleic acid sequence comprises a label that may be the same or a different label. When primer P1 used in the amplification of the target nucleic acid sequence comprises a label, primer P3 comprises a label that may be the same or a different label. Primer P1 is extended by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex Bxe2x80x2. The tailed target partial duplex Axe2x80x2 is allowed to bind to the tailed reference partial duplex Bxe2x80x2 to form a complex. The binding of one of the labels to another of the labels as a result of the formation of the complex is detected. Such binding is directly related to the presence of the target nucleic acid sequence.
Another aspect of the present invention is a method for detecting a target nucleic acid sequence wherein a partial duplex Axe2x80x2 is produced from a target nucleic acid sequence. The duplex Axe2x80x2 comprises a fully complementary double stranded nucleic acid sequence containing the target nucleic acid sequence wherein one strand has at its 5xe2x80x2-end a portion A1 that does not hybridize with a corresponding portion A2 at the 3xe2x80x2-end of the other strand. At least one of the strands of the partial duplex Axe2x80x2 comprises a label. A partial duplex Bxe2x80x2 is produced from a reference nucleic acid sequence and comprises a double stranded nucleic acid sequence different from the target nucleic acid sequence. The strand corresponding to the strand comprising portion A1 has at its 5xe2x80x2-end a portion B1 that is complementary with A2 and the other strand has at its 3xe2x80x2-end a portion B2 that is complementary with Al. One of the strands of partial duplex Bxe2x80x2 comprises a label wherein such strand is unable to hybridize directly to the strand of partial duplex Axe2x80x2 that comprises a label. Partial duplexes Axe2x80x2 and Bxe2x80x2 are subjected to conditions that permit the duplexes to hybridize to each other to form a stable quadramolecular complex. A determination is made as to whether such complex is formed. The presence thereof indicates the presence of the target nucleic acid sequence. In an alternate embodiment the strands comprising A1 and B1 have labels instead of the strands comprising A2 and B2.
Another embodiment of the present invention is a method for detecting the presence of a difference between two related nucleic acid sequences wherein a target nucleic acid sequence and a reference nucleic acid sequence are produced from the two related nucleic acid sequences. Each respective strand of the target nucleic acid sequence and the reference nucleic acid sequence produced has a portion introduced therein that is a nucleotide sequence priming site. A partial duplex Axe2x80x2 is produced from the target nucleic acid sequence using the nucleotide sequence priming sites. Partial duplex Axe2x80x2 comprises a fully complementary double stranded nucleic acid sequence containing the target nucleic acid sequence wherein one strand has at its 5xe2x80x2-end a portion A1 that does not hybridize with a corresponding portion A2 at the 3xe2x80x2-end of the other strand. One of the strands of the partial duplex Axe2x80x2 may comprise a label. A partial duplex Bxe2x80x2 is also produced from the reference nucleic acid sequence using the nucleotide sequence priming sites. Partial duplex Bxe2x80x2 comprises the double stranded nucleic acid sequence wherein the strand corresponding to the strand comprising portion A1 has at its 5xe2x80x2-end a portion B1 that is complementary with A2 and the other strand has at its 3xe2x80x2-end a portion B2 that is complementary with A1. One of the strands of the partial duplex Bxe2x80x2 may comprise a label wherein a strand comprising a label is unable to hybridize directly to a strand of the partial duplex Axe2x80x2 when that strand comprises a label. The partial duplexes Axe2x80x2 and Bxe2x80x2 are subjected to conditions that permit the duplexes to hybridize to each other. If the related nucleic acid sequences have a difference, a stable complex is formed comprising partial duplex Axe2x80x2 and partial duplex Bxe2x80x2. A determination is made as to whether a stable complex is formed, the presence thereof indicating the presence of a difference between the two related nucleic acid sequences.
Another embodiment of the present invention is a method for detecting the presence of a mutation in a target nucleic acid sequence comprising amplification of target and reference nucleic acid sequences by polymerase chain reaction using primers PX1i and PX2i to produce a target sequence or a reference sequence, respectively, comprising nucleotide sequence priming sites Paxe2x80x2 and P2xe2x80x2. The reference sequence is identical to the target sequence but lacks a possible mutation. The target sequence produced above is amplified by polymerase chain reaction, using primers P1 and P2, to produce an amplicon AA. One of primers P1 and P2 may comprise a label and primer P1 is comprised of a 3xe2x80x2-end portion Pa that can hybridize with priming site Paxe2x80x2 of the target sequence and 5xe2x80x2-end portion B1 that cannot hybridize with the target sequence. A primer P3 is extended by chain extension along one strand of amplicon AA to produce a tailed target partial duplex Axe2x80x2. Primer P3 is comprised of 3xe2x80x2-end portion Pa and a 5xe2x80x2-end portion A1 that cannot hybridize to the target sequence or its complement. The reference sequence produced above is amplified, using primer P2 and primer P3, by polymerase chain reaction to produce amplicon BB. Primer P2 may comprise a label when primer P2 above comprises a label and primer P3 may comprise a label when primer P1 above comprises a label. Primer P1 is extended by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex Bxe2x80x2. The tailed target partial duplex Axe2x80x2 is allowed to bind to the tailed reference partial duplex Bxe2x80x2. The formation of a stable complex between the tailed partial duplexes is detected, the formation thereof being directly related to the presence of the mutation.
Another embodiment of the invention is a kit as mentioned above that also includes a pair of adapter primers for amplifying the target and reference nucleic acids. One of the primers has a 3xe2x80x2-end portion that is hybridizable to the target and reference nucleic acids and a portion 5xe2x80x2 thereof that is not hybridizable with the target or reference nucleic acids and is substantially identical to primer P2. The other of the primers has a 3xe2x80x2-end portion that is hybridizable to the target and reference nucleic acids and a portion 5xe2x80x2 thereof that is not hybridizable with the target or reference nucleic acids and is substantially identical to the 3xe2x80x2-end portion Pa of primers P1 and P3. The adapter primers are usually packaged in a container separate from primers P1, P2 and P3.